Gene therapy has been widely used in clinical trials since 1990s with many successful cases reported using viral or non-viral vectors to deliver therapeutic genes. The lung is an important organ for the gene therapy treatment to patients with inherent gene defects such as cystic fibrosis (CF), alpha 1-antitrypsin (AAT) deficiency, or with other chronic acquired respiratory disorders such as asthma and lung cancers. Of these lung diseases, CF, caused by single gene defect in coding a protein cystic fibrosis transmembrane conductance regulator (CFTR), is the most common life-threatening gene defect inherent disease with about $450 million spent annually on patient care in the U.S alone. Although clinical treatments have improved CF patients' quality of life and lifespan in the recent decades, for this single gene defect inherent disease, gene therapy appears the best cure to permanently correct the disorder by replacing the defective CFTR gene (Mueller et al., 2008; Driskell et al., 2003; Griesenbach et al., 2010).
CF is an autosomal recessive genetic disorder caused by mutations in the CFTR gene coding (Rommens et al., 1989). It is a multi-organ disease, but CF pulmonary disease is the most life-threatening (Rowe et al., 2005). Recombinant adeno-associated viral vectors (rAAV) are currently one gene therapy agent that is being pursued for CF lung gene therapy (Griesenbach et al., 2010; Flotte, 2007; Carter, 2005).
rAAV vectors for CF lung gene therapy have been under development for nearly two decades, and most serotypes appear to be effectively endocytosed from the apical surface of airway epithelia despite varying degrees of transduction (i.e., expression of an encoded transgene). Although these vectors have demonstrated good safety profiles in CF clinical trails (Aitken et al., 2001; Moss et al., 2007; Wagner et al., 2002), they have failed to achieve complementation in vivo for two significant reasons. First, post-entry barriers in virion processing following infection appear to limit nuclear translocation, and thus transgene expression, in a proteasome-dependent manner (Duan et al., 2000; Ding et al., 2005; Yan et al., 2002; Zhong et al., 2008; Zhong et al., 2007). This feature of rAAV2 is reflected in CF clinical trials where viral genomes persisted in the airway epithelia of test subjects without detection of transgene-derived CFTR mRNA or clinical improvement in lung function (Aitken et al., 2001; Moss et al., 2007; Wagner et al., 2002). Identifying an appropriate rAAV serotype that bypassed these limitations has proved challenging due to species-specific differences between animal models and humans (Flotte et al., 2010; Liu et al., 2007a; Liu et al., 2007b). rAAV1 proves to be the most efficient serotype for apical infection of human airway (Flotte et al., 2010; Yan et al., 2012; Yan et al., 2006), while others have found success using directed capsid evolution to enhanced the tropism of rAAV for apical human epithelium (HAE) transduction (Li et al., 2009; Excoffon et al., 2009). However, effective CFTR complementation in CF HAE still requires the use of proteasome inhibitors to enhance transduction (Li et al., 2009; Zhang et al., 2004).
A second major barrier to efficient CFTR expression from rAAV vectors is their limited packaging capacity (about 4.9 kb) that necessitates the use of small, weak promoters and/or the use of CFTR minigenes (Zhang et al., 1998). The first generation rAAV-CFTR tested in a clinical trial utilized the cryptic promoter within the AAV2 ITR to drive the expression of a full-length CFTR cDNA (Aitken et al., 2001), and this was later improved by the incorporation of a short 83 bp synthetic promoter (Zhang et al., 2004). Other efforts to circumvent the small packing capacity of rAAV vectors have included trimming down size of the CFTR cDNA by deletion of non-critical sequences (such as partial deletion at the R-domain) to expand room for core promoter elements such as a shortened CMV promoter (Li et al., 2009; Zhang et al., 1998; Ostedgaard et al., 2005; Ostedgaard et al., 2002). Although these strategies have improved expression of CFTR, it is clear that pushing the packaging limits of rAAV can lead to inconsistent deletions at the 5′ end of rAAV genome (Kapranov et al., 2012), thus further jeopardizing genome stability and expression.